ABSTRACT
The proliferation of vascular smooth muscle cells (SMCs) is a key event in the development of atherosclerotic lesions and in the restenosis of arteries after angioplasty. Polypeptide growth factors are potent SMC mitogens in vitro and are believed to be involved in SMC proliferation in vivo. Strong data exist linking platelet-derived growth factor (PDGF) activity to human atherosclerosis. However, no low-molecular-weight antagonists of this growth factor have been discovered. We identified a compound, SCH 13929 (2-bromomethyl-5-chlorobenzene sulfonylphthalimide), which inhibits binding of 125I-PDGF BB to cell surface receptors with an IC50 of 0.13 mumol/L. This compound has a lesser effect on the binding of 125I-epidermal growth factor (EGF), 125I-basic fibroblast growth factor (bFGF), or 125I-endothelin to specific cell surface receptors. Exposure of cultured SMCs to SCH 13929 inhibits PDGF BB- and PDGF AA-stimulated DNA synthesis but not EGF- or bFGF-stimulated DNA synthesis. PDGF BB-stimulated SMC division is also inhibited by exposure to SCH 13929. Chemotaxis assays indicate that SCH 13929 inhibits PDGF-stimulated directional migration and suggest that the compound interacts with PDGF rather than with the receptor. Oral administration of SCH 13929 (100 mg/kg per day) to Sprague-Dawley rats or spontaneously hypertensive rats results in significant inhibition of lesion formation in the balloon catheter-deendothelialized carotid artery. These results suggest that SCH 13929 may be a useful tool for understanding the role of PDGF in intimal lesion formation.
Subject(s)
Chlorobenzenes/pharmacology , Phthalimides/pharmacology , Platelet-Derived Growth Factor/antagonists & inhibitors , Receptors, Platelet-Derived Growth Factor/antagonists & inhibitors , Tunica Intima/metabolism , 3T3 Cells/drug effects , Animals , Cattle , Cell Division , Cells, Cultured , Chemotaxis, Leukocyte/drug effects , DNA/biosynthesis , Fibroblasts/drug effects , Growth Substances/pharmacology , Humans , Hypertension/pathology , Male , Mice , Mice, Inbred BALB C , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/metabolism , Platelet-Derived Growth Factor/metabolism , Rats , Rats, Inbred SHR , Rats, Sprague-Dawley , Receptors, Platelet-Derived Growth Factor/metabolism , Tunica Intima/pathologyABSTRACT
Human interleukin 4 is a highly pleiotropic cytokine secreted by activated T cells that exerts multiple biological effects on B and T lymphocytes and other cell types. Elucidation of structure-function relations was accomplished by epitope mapping of a panel of monoclonal antibodies and by mutagenesis of selected amino acid residues. Epitope mapping of these monoclonal antibodies was achieved through binding studies with recombinant human interleukin 4 (rhuIL-4), proteolytic fragments produced by digestion with Staphylococcus aureus V8 protease and synthetic peptides derived from the sequence of the parent molecule. Monoclonal antibodies 25D2, 35F2, and 11B4 neutralized the in vitro T-cell proliferation activity of rhuIL-4 and also prevented binding of rhuIL4 to its cell surface receptor. These antibodies recognized sequences 104-129, 70-92, and 61-82, respectively. These regions comprise the BC loop/helix C (residues 61-92) and helix D (residues 104-129). A nonneutralizing monoclonal antibody (1A2) recognized a nonoverlapping region (residues 43-59) comprising almost entirely helix B. Mutagenesis of a cluster of residues within helix C showed that at least three residues (K84, R88, and N89) were potentially involved in receptor recognition. The existence of two distinct nonneighboring binding domains in the three-dimensional structure of rhuIL-4 provided preliminary evidence for a model of receptor interaction involving the formation of a ternary complex consisting of two molecules of the extracellular portion of the receptor and one molecule of rhuIL-4.
Subject(s)
Epitopes/chemistry , Interleukin-4/chemistry , Peptide Mapping , Antibodies, Monoclonal , Blotting, Western , Humans , Interleukin-4/genetics , Interleukin-4/immunology , Models, Molecular , Molecular Structure , Mutagenesis , Peptide Fragments/chemistry , Peptide Fragments/immunology , Peptide Fragments/metabolism , Protein Structure, Secondary , Recombinant Proteins/metabolism , Serine Endopeptidases/metabolism , Structure-Activity Relationship , X-Ray DiffractionABSTRACT
Deletion of nine amino acids from the carboxyl terminus of human IFN gamma (residues 138--146; LFRGRRASQ) resulted in a 7-fold increase in specific antiviral activity. Similar increases in receptor binding affinity were seen. Deletion of residues 136 and 137 (QM) had little additional effect, but removal of Ser135 resulted in a sharp drop in antiviral activity. Further removal of residues 133 and 134 (KR) lowered antiviral activity to 1% of the peak value. Comparison of the proton NMR spectra of selected deletions down to residue 132 showed that there was no significant change in the core protein structure. Deletions down to residue 125 had the same antiviral activity as those to 132, but changes could now be seen in the aromatic proton NMR spectrum of this shorter derivative. Substitution of the homologous murine sequence between residues 124 and 130 (human SPAAKTG; murine LPESSLR) resulted in only a small decrease in antiviral activity, further suggesting that the precise sequence in this region was not critical for activity. Ser135 was substituted with a number of other amino acids with little or no change in activity. The importance of the residues between 131 and 134 for biological activity was corroborated by mutagenesis, although some substitutions in this region were tolerated.
Subject(s)
Antiviral Agents/chemistry , Interferon-gamma/chemistry , Serine/chemistry , Animals , Base Sequence , Binding Sites , Chromosome Deletion , Humans , Interferon-gamma/genetics , Interferon-gamma/physiology , Magnetic Resonance Spectroscopy , Mice , Molecular Sequence Data , Mutagenesis, Site-Directed , Receptors, Immunologic/physiology , Receptors, Interferon , Structure-Activity RelationshipABSTRACT
Human IL-4 (hIL-4) has been cloned from a human T cell line based on its homology to the murine IL-4 cDNA sequence. We have compared cytoplasmic and extra-cytoplasmic expression of this basic protein in Escherichia coli using various combinations of promoters, replicons and host strains. Strains producing a cytoplasmic product were most successful at heterologous protein expression, producing up to 500 mg/l of an inactive aggregated form of the protein. The biological activity of the protein could be restored by refolding the protein with guanidine hydrochloride and glutathione giving a specific activity identical to that of IL-4 derived from CHO cell lines stably transformed with an hIL-4 expression plasmid. Strains designed to secrete human IL-4 into the periplasmic space produced far less protein (approximately 5 mg/l). However, a significant fraction of this protein was detected in the culture medium. This fraction appeared to be soluble after ultracentrifugation, and demonstrated high specific activity without refolding. Leakage of heterologous protein into the culture medium may be a viable way to recover biologically active products without relying on the denaturation and refolding in vitro that can, at times, yield incorrectly folded gene product.
Subject(s)
Escherichia coli/genetics , Interleukin-4/biosynthesis , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Cytoplasm/metabolism , Electrophoresis, Polyacrylamide Gel , Escherichia coli/metabolism , Humans , Interleukin-4/genetics , Interleukin-4/metabolism , Molecular Sequence Data , Plasmids , Protein Conformation , Solubility , Species SpecificityABSTRACT
The purified outer membrane from F- (W1-3) cells was shown to inhibit mating effectively, but the purified cytoplasmic (inner) membrane did not. These membranes, heat-treated minicells, and ultraviolet-irradiated minicells were examined for their ability to generate a mating signal at 43 degrees C in mating with HfrH dnaB(Ts) cells. The outer and inner membranes and heat-treated minicells all failed to stimulate incorporation of radioactive thymine; only ultraviolet-irradiated minicells retained the ability to generate a mating signal for the donor to initiate transfer replication.
Subject(s)
Escherichia coli/genetics , F Factor , Cell Membrane/physiology , Conjugation, GeneticABSTRACT
The addition of 1 mM orthophenanthroline (OP) into a mating mixture drastically reduced the production of recombinants. Examination of the effect of OP on each step of conjugation showed that the effect on the following steps could not account for the up to 500-fold reduction of recombinant formation: (i) preliminary mate formation and (ii) deoxyribonucleic acid transfer and integration. Taking these results and additional experiments together, we conclude that OP inhibits the maturation of preliminary mates into effective mates. Kinetic experiments showed that there were two phases in the maturation of preliminary (OP-sensitive) mates into effective (OP-resistant) mates. The half-time (the time required to reach 50% OP-resistant mates) was 2.5 min for the first phase and 4 min for the second phase, with an overall half-time of 7.5 min. In contrast, only 3 min was required to reach 50% Zn2+-resistant mates. The difference in half-time suggests that there is an intermediate step involved to form an effective mate from a preliminary mate.